Oligosaccharide synthesis

ABSTRACT

The invention provides a system for solid-phase synthesis of oligosaccharides, based on the discovery that a 2-substituted-1,3-dioxocycloalkyl linker group of general formula (I) can be used to couple saccharide groups of both the O-glycoside and N-glycoside type to a polymer support. The invention provides reagents, reagent kits and methods for solid-phase oligosaccharide synthesis.

FIELD OF THE INVENTION

[0001] This invention relates to methods for synthesis ofoligosaccharides, and in particular to methods for solid phase orcombinatorial synthesis of oligosaccharides. The invention provides anovel linker-resin, linker-saccharide, or resin-linker-saccharidecomplex, which in one embodiment enables a saccharide residue to belinked to a soluble or insoluble polymeric support for use as a basisfor solid-phase synthesis of oligosaccharides. In a second embodiment,the complex of the invention enables oligosaccharides to be linked to asolid polymeric support for use as an analytical reagent.

BACKGROUND OF THE INVENTION

[0002] Oligosaccharides constitute a major class of bioactive polymers,implicated in biochemical processes (Lasky, 1992; Varki, 1993) asdiverse as cellular differentiation, hormone-cell recognition andcell-cell adhesion, especially viral-host cell (Gambaryan et al, 1995)and bacteria-host cell attachment (Boren et al, 1993). Involvement ofoligosaccharides in diseases such as cancer, cardiovascular disorders,microbial infections, graft rejection and autoimmune disorders hastherefore, been strongly suggested. Conjugation of carbohydrates tobioactive peptides has also been demonstrated to stabilise the peptidesagainst degradation, and, in more specific circumstances, to facilitatepeptide transport across biological barriers (Lee, 1989; Fisher, 1991;Rodriguez, 1989). Thus the ability to synthesise oligosaccharides in afacile and efficient manner is now becoming an extremely important areawithin organic chemistry.

[0003] The highly labour intensive solution phase strategies hithertoutilised in oligosaccharide syntheses require an extremely specialisedknowledge and a high degree of chemical skill. This situation wasmirrored within the area of peptide synthesis, until Merrifield et alproposed and developed Solid Phase Peptide Synthesis (SPPS) over thirtyyears ago (Merrifield, 1963). In SPPS immobilisation of the first aminoacid of the required sequence to an insoluble resin enabled largeexcesses of reagents to be used to achieve the coupling of the secondamino acid. Any unused materials remaining at the end of the couplingstep could then be removed simply by washing the resin beads. Thistechnology meant that the chemist could drive each coupling reaction toalmost quantitative yields, and since the peptide intermediates formedwere still bound to the resin, purification after each acylation stepwas not required. SPPS enables peptide and polypeptide synthesis to beemployed as a routine research and synthetic tool, and permitslarge-scale combinatorial synthesis of peptides for screening ofpotential pharmaceutical agents.

[0004] For many years chemists have attempted to transpose thissolid-phase methodology to oligosaccharide synthesis, with varyingdegrees of success. The first attempt was approximately 25 years ago(Frechet and Schuerch, 1971; Frechet and Schuerch, 1972; Guthrie et al,1971; Guthrie et al, 1973). However, the ozone-mediated deprotectionproduct was an aldehyde-substituted glycoside. Danishefsky and coworkersdescribed the solid phase synthesis of the Lewis b Antigen (Randolph etal, 1995) and N-linked glycopeptides (Roberge et al, 1995) by initialattachment of the primary sugar unit of the oligosaccharide to a 1%divinylbenzene-styrene co-polymer support via a silyl ether linkage. Theresin-bound sugar moeity was in this instance a glycal, with on-resinactivation achieved via epoxidation of the double bond, and theresulting glycal residue acting as a sugar donor through nucleophilering-opening of the epoxide. Since there are no calorimetric methodsavailable to the sugar chemist to monitor on-resin glycosylations, theonly means of assessing the progress of the reaction is by lysis of theoligosaccharide-resin bond and subsequent analysis of the cleavageproduct, usually by thin layer chromatography. The tetra-n-butylammoniumfluoride-mediated deprotection conditions required to cleaveDanishefsky's silyl ether linker are both hazardous and slow. Thiscoupled with the requirement for on-resin activation of the tetheredglycals, makes the overall strategy and methodology far from ideal.

[0005] In an alternative approach, Douglas and coworkers described thesynthesis of D-mannopentose using a polyethyleneglycol ω-monomethyletherco-polymer and a succinoyl or an α,α′-dioxyxylyl diether linker (Douglaset al, 1995). The reactions were carried out in solution phase, withremoval of unused reactants being achieved by precipitation of theoligosaccharide-polymer complex and subsequent washing. In the latterexample, cleavage of the oligosaccharide-polymer bond was achievedthrough catalytic hydrogenation, which required exposure of theconjugate to 1 atm of H₂ for 48 h to achieve respectable yields. Thisagain is far too slow to allow effective monitoring of individualglycosylation reactions. Yan et al reported sulphoxide-mediatedglycosylation on a Merrifield resin, using a thiophenol linker for theattachment of the primary sugar residue (Yan et al, 1994). This methodresulted in the construction of (1-6)-linked oligosaccharides, and wassuitable for synthesis of both α- and β-glycosidic linkages. However,the thioglycosidic linkage to the resin dictates that similar sugardonors cannot be employed in this strategy.

[0006] Recently Rademann and Schmidt reported the use oftrichloroacetimidate sugar donors to a resin bound sugar tethered via analkyl thiol (Rademann and Schmidt, 1996); once again, however, thismethod precludes the use of the far superior thioglycoside sugar donors.Meanwhile, Adinolfi et al described the synthesis of disaccharides usinga polyethyleneglycol-polystyrene resin, with connection of the firstsugar to the polymeric support through a succinate spacer (Adinolfi etal, 1996). However, the acid lability displayed by this linker meansthat the primary sugar cannot be linked to the resin via the glycosidicposition.

[0007] The above examples serve to illustrate that the critical elementin solid phase synthesis is the nature of the linker between the solidsupport and the initial synthon. The linker must display excellentstability to the conditions of coupling and deprotection, yet in thecase of solid phase oligosaccharide synthesis, it should also be rapidlyand efficiently cleaved to allow monitoring of the progress ofindividual coupling reactions. The cleavage should ideally be achievedby the use of a relatively innocuous chemical reagent.

[0008] It is clear, then, that there remains a need in the art forsimple, efficient and economical methods for solid-phase synthesis ofoligosaccharides.

[0009] A hydrazine-labile primary amino-protecting group,N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde), has beenreported for protection of lysine side chains during SPPS (Bycroft etal, 1993). This group was modified for use as a carboxy-protecting groupin SPPS when the 2-(3-methylbutyryl)dimedone analogue of2-acetyl-dimedone was condensed with 4-aminobenzylalcohol to afford4-[N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methyl-butyl]-amino]benzylester (ODmab)(Chan et al, 1995).

[0010] The two protecting groups were reported to be stable to thedeprotecting conditions widely used in SPPS, ie. trifluoroacetic acid(TFA) or 20% piperidine in dimethyl formamide (DMF). The ethyl ester,4-[N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)aminolbenzyl ester(ODab) showed small but significant instability to 20% piperidine-DMF.Both Dde and ODmab are linked to groups on amino acids, rather thandirectly to the solid-phase support. Their use in solid-phaseoligosaccharide synthesis has not been suggested.

[0011] We have now surprisingly found that protecting groups similar toDde and ODmab can be coupled to a polymeric support, thereby generatinga system for the immobilisation of sugars. To this end we haveimmobilised N- and O-glycosides to the solid support and synthesisedoligosaccharides using various sugar donors. The linkers displayexcellent stability to most acids and secondary/tertiary basesencountered in modern synthetic chemistry, yet are rapidly andefficiently cleaved with either ammonia, hydrazine or primary amines.

[0012] Bannwarth et al have independently developed a different solidphase linker around the Dde protecting group, which they have utilisedfor the immobilisation of amino acids and primary amines forcombinatorial library synthesis (Bannwarth et al, 1996). However, thesynthesis of this linker is both protracted and inefficient, and thelinker only displays a limited stability to secondary bases such aspiperidine. There has been no suggestion that this linker could be usedfor oligosaccharide synthesis. The linkers we have developed demonstratea far greater stability than those of Bannwarth et al.

SUMMARY OF THE INVENTION

[0013] In one aspect, the invention provides a support for solid-phasesynthesis of oligosaccharides, said support comprising:

[0014] a) a resin,

[0015] b) a linker covalently attached to the resin, and

[0016] c) one or more saccharide groups covalently attached to the resinvia the linker,

[0017] wherein the linker is a 2-substituted-1,3-dioxocycloalkanecompound, and

[0018] said support having general formula I:

[0019] in which

[0020] R¹ and R² may be the same or different, and is each hydrogen orC₁₋₄ alkyl;

[0021] R′ is an amino sugar, a glycosylamine, or a glycosylamine of anoligosaccharide; a mono or oligosaccharide coupled through an alkyl-,substituted alkyl-, aryl-, substituted aryl-, cycloalkyl-, orsubstituted cycloalkyl-amino group; or a mono or oligosaccharide coupledthrough a carboxyalkyl-, substituted carboxyalkyl-, carboxyaryl-,substituted carboxyaryl-, carboxycycloalkyl-, or substitutedcarboxycycloalkyl-amino group; and

[0022] R″ is an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, or substituted cycloalkyl spacer group which is directlycoupled to the resin support, or which may optionally be coupled to theresin support via a suitable covalent linkage, which is stable toconditions of oligosaccharide synthesis and cleavage.

[0023] The covalent linkage to the resin may suitably be provided by a—CONH—, —O—, —S—, —COO—, —CH═N—, —NHCONH—, —NHCSNH, or —NHNH— grouping,eg. Spacer-CONH-resin, Spacer-O-resin, Spacer-S-resin, Spacer-CO₂-resin,Spacer-CH═N-resin, Spacer-NHCONH-resin, Spacer-NHCSNH-resin,Spacer-NHNH-resin. Other possible covalent linking groups will be knownto those skilled in the art.

[0024] Preferably both R¹ and R² are methyl.

[0025] Preferably R′ is an oligosaccharide-O—CH₂—(C₆H₄)—NH,monosaccharide-O—CH₂—(C₆H₄)—NH, amino-oligosaccharide-CO₂CH₂—(C₆H₄)NH,or amino-monosaccharide-CO₂CH₂—(C₆H₄)—NH group.

[0026] In a particularly preferred embodiment the2-substituted-1,3-dioxocycloalkane linker is functionalised Dde, Ddh orODmab. In one very particularly preferred embodiment the supportcomprises a resin, a linker and a monosaccharide, an oligosaccharide, anaminosaccharide or an amino-oligosaccharide.

[0027] In a second aspect, the invention provides a support forsolid-phase synthesis comprising a resin and a linker group, wherein thelinker is a 2-substituted-1,3-dioxocycloalkane of general formula II:

[0028] in which

[0029] X is OH or NH_(2;)

[0030] R¹ and R² may be the same or different, and is each hydrogen orC₁₋₄ alkyl; preferably both R¹ and R² are methyl; and

[0031] R″ is an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, or substituted cycloalkyl spacer group which is directlycoupled to the resin support, or which may optionally be coupled to theresin support via a suitable covalent linkage, which is stable toconditions of oligosaccharide synthesis and cleavage. The covalentlinkage may suitably be provided by a —CONH—, —O—, —S—, —COO—, —CH═N—,—NHCONH—, —NHCSNH, or —NHNH— grouping, eg. Spacer-CONH-resin,Spacer-O-resin, Spacer-S-resin, Spacer-CO₂-resin, Spacer-CH═N-resin,Spacer-NHCONH-resin, Spacer-NHCSNH-resin, Spacer-NHNH-resin. Otherpossible covalent linking groups will be known to those skilled in theart.

[0032] In a third aspect, the invention provides a linker-saccharidecomplex, comprising a linker group of general formula II as definedabove and a saccharide group as defined above for R′.

[0033] In a fourth aspect the invention provides a linker compoundcarrying functional groups suitable to attach a primary amine to a resinvia covalent bonds which are stable to conditions of oligosaccharidesynthesis and cleavage, said compound having general formula III

[0034] in which

[0035] X is OH or NH₂;

[0036] R¹ and R² may be the same or different, and is each hydrogen orC₁₋₄ alkyl, and

[0037] R″ is an alkyl, substituted alkyl, aryl, substituted aryl,cycloalkyl, or substituted cycloalkyl spacer group, which carries afunctionality capable of reacting with a functionalised resin.

[0038] Preferably the linker compound is6-hydroxyl-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid or anester thereof. Preferably the ester is a benzyl, methyl, or t-butylester.

[0039] For the purposes of this specification the term “substituted” inthe definitions of substituents within this specification means that thesubstituent is itself substituted with a group which does not change thegeneral chemical characteristics of the substituent. Preferred suchfurther substituents are halogen, nitro, amino, hydroxyl, and thiol;preferred halogens are chlorine and iodine. The person skilled in theart will be aware of other suitable substituents of similar size andcharge characteristics which could be used as alternatives in a givensituation.

[0040] For the purposes of this specification a compound is regarded as“stable to conditions of oligosaccharide synthesis and cleavage” ifthere is less than 10% loss of the compound after exposure at roomtemperature to ammonia, hydrazine or a primary amino compound in wateror DMF. The person skilled in the art will readily be able to determinewhether the stability of a particular compound is adequate for it to beuseful for the purposes of the invention, using conditions appropriatefor his or her particular needs.

[0041] The linker compound of the invention may be synthesized on theresin, or may be synthesized in solution.

[0042] The invention also provides kits useful in solid phase synthesisor combinatorial synthesis of oligosaccharides, comprising either

[0043] a) a resin-linker-saccharide support,

[0044] b) a linker-saccharide complex, or

[0045] c) a resin-linker support, according to the invention, asdescribed above. The kit may optionally also comprise one or morefurther reagents such as protecting agents, deprotecting agents, and/orsolvents suitable for solid phase or combinatorial synthesis. The personskilled in the art will be aware of suitable further reagents. Differenttypes of kit can then be chosen according to the desired use.

[0046] The resin may be any resin which swells in water and/or in anorganic solvent, and which comprises one of the following substituents:halogen, hydroxy, carboxyl, SH, NH_(2,) formyl, SO₂NH_(2,) or NHNH_(2,)for example methylbenzhydrylamine (MBHA) resin, amino or carboxytentagel resins, 4-sulphamylbenzyl AM resin. Other suitable resins willbe known to those skilled in the art.

[0047] The invention also provides a method of solid-phase synthesis ofoligosaccharides, comprising the step of sequentially linking mono- oroligosaccharide groups to a support as described above. Similarly themono- or oligosaccharide building blocks may be as described above.

[0048] This method is particularly useful for combinatorial syntheticapplication.

[0049] The linker compound may be synthesised in solution or directly onthe resin in a stepwise manner prior to the coupling of the initialsugar group, or the linker-initial sugar conjugate may be synthesised insolution phase and subsequently coupled to the solid support, withsubsequent sugars being sequentially attached. Preferably the second andall subsequent sugar groups are coupled to the oligosaccharidechain-resin conjugate after the last sugar in the oligosaccharide chainis partially deprotected.

[0050] The invention accordingly provides a method of synthesis of alinker group according to general formula I as defined above, comprisingthe step of C-acylation of a 2-substituted 1,3-dioxocyclohexane compoundwith a dicarboxylic acid. Preferably the dicarboxylic acid ismono-protected by ester formation. More preferably the reaction isactivated with carbodiimide and catalysed by N,N′-dimethylaminopyridine.

[0051] The product of the reaction may optionally be reacted with4-aminobenzyl alcohol, to form the 4-aminobenzyl derivative.

[0052] The invention also provides a method of synthesis of aresin-linker support, comprising the step of swelling a resin in asuitable solvent, treating the swollen resin with a dicarboxylic acid,and reacting the thus-produced product with a 2-substituted1,3-dioxocycloalkane compound. Preferably for both synthesis of thelinker and synthesis of the resin-linker support the 2-substituted1,3-dioxocyclolkane compound is 5,5-dimethyl-1,3-cyclohexanedione. Alsopreferably the dicarboxylic acid is adipic acid.

[0053] The first sugars attached to the resin-linker unit may beunprotected, partially protected or fully protected glycosides,aminoglycosides, or ether- or amino-linked sugars, where the couplingtakes place through a non-glycosidic position.

[0054] The building block mono- or oligosaccharide-donors may be anyactivated sugar, including but not limited to orthoesters,thioorthoesters, cyanoalkylidene derivatives, 1-O-acyl sugars, aminosugars, acetimidates, trichloroacetimidates, thioglycosides,aminoglycosides, amino-oligosaccharides, glycosylamines ofoligosaccharides, glycosyl thiocyanates, pentenyl glycosides,pentenoylglycosides, isoprenyl glycosides, glycals, tetramethylphosphorodiamidates, sugar diazirines, selenoglycosides, phosphorodithioates,glycosyl-dialkylphosphites, glycosylsulphoxides and glycosylfluorides.

[0055] Preferably the first sugar coupled to the resin is an aminosugar,an aminoglycoside, or an amino-oligosaccharide or a glycosyl amine of anoligosaccharide.

[0056] Preferably partial sugar deprotection is achieved by usingacyl-type, trityl, benzyl-type, acetal-type, or various silyl and/orphotolabile protecting groups in addition to permanent protectinggroups. This permits the synthesis of branched oligosaccharides by usingtwo orthogonal hydroxy-protecting groups on a single sugar donor.

[0057] The synthesised oligosaccharide can be cleaved from the resinusing ammonia, hydrazine or a primary amine, such as butylamine orcyclohexylamine. For the preparation of aminoglycosides, ammonia or asuitable primary amine in an organic solvent is preferably employed. Forthe preparation of hydrazides, hydrazine in water or in an organicsolvent is preferably employed. For the preparation of oligosaccharides,ammonia in water or in an organic solvent is preferably employed,followed by acidification. When the linker contains a 4-aminobenzylmoiety, after cleavage as described above the first sugar is releasedstill protected by the aminobenzyl group; this can be removed byhydrogenation if desired.

[0058] The person skilled in the art will appreciate that theoligosaccharide can be retained on the resin for use as an analytical orpreparative reagent, for example in affinity chromatography or forbulk-scale affinity separation.

DETAILED DESCRIPTION OF THE FIGURES

[0059]FIG. 1 shows a general representation of the strategy required forsolid phase oligosaccharide synthesis.

[0060]FIG. 2 illustrates a general representation of the‘divide-couple-recombine’ method of oligosaccharide library synthesisutilising a solid phase strategy.

[0061]FIG. 3 shows the synthesis of the Dde-based linker of theinvention, attachment of the primary sugar residue and coupling of thesugar-linker conjugate to a resin support. An alternative approachwhereby the linker is synthesised directly on the resin is also shown.

[0062]FIG. 4 shows the synthesis of the ODmab-based linker of theinvention, attachment of the primary sugar residue and coupling of thesugar-linker conjugate to the resin support.

[0063]FIG. 5 shows the cleavage of the oligosaccharide-linker bond in aresin-bound hydrazine mediated deprotection product.

[0064]FIG. 6 shows a general representation of the selectivedeprotection of one sugar hydroxyl group, and subsequent coupling of thenext sugar donor.

[0065]FIG. 7 shows the immobilisation of an amino-oligosaccharide on theDde-derivatised support.

[0066]FIG. 8 shows a list of activated sugar donors for solid-phasesynthesis.

[0067]FIG. 9 shows the synthesis of a differentially protectedthioglycoside and a partially protected aminoglycoside.

[0068]FIG. 10 shows the trichloroacetimidate activation of the4-aminobenzyl modified linker.

[0069]FIG. 11 shows ammonia-mediated cleavage of the aminoglycoside withpost-cleavage acidification to generate the free carbohydrate.

[0070]FIG. 12 shows a specific example of the general strategy foroligosaccharide synthesis employing a thiogycoside as the sugar donor.

[0071]FIG. 13 shows another specific example of the general strategy foroligosaccharide synthesis employing a thiogycoside as the sugar donor.

[0072]FIG. 14 shows the cleavage of a monosaccharide bound to the4-aminobenzyl modified linker.

[0073]FIG. 15 shows an example of a resin-bound fully protectedtrisaccharide.

[0074]FIG. 16 shows the immobilisation of an unprotected amino sugar.Detailed Description of the Invention Abbreviations used herein are asfollows: Bn Benzyl Bu Butyl DCM Dichloromethane DdeN-1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl Ddh-OH6-Hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene) hexanoic acid DMAPN,N′-Dimethyl aminopyridine DMF N,N′-Dimethylformamide DMTSTDimethyl(methylthio)sulphonium trifluoromethanesulphonate EEDQ1-Isobutyloxycarbonyl-2-isobutyloxy-1,2- dihydroquinoline EtOAc Ethylacetate EtOH Ethanol FAB-MS Fast atom bombardment mass spectrometry HRMSHigh resolution mass spectrometry MBHA Methyl benzyhydrylamine resin MeMethyl MeOH Methanol NMR Nuclear magnetic resonance ODmab4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]-amino}benzyl alcohol. PEG Polyethylene glycol tButetra-butyl TFA Trifluoroacetic acid THF Tetrahydrofuran TLC Thin-layerchromatography TNBS 2,4,6-Trinitrobenzene sulphonic acid

[0075] The invention is based upon the immobilisation of a Dde-, Ddh orODmab-based linker to a polymer support in order to tether anysaccharide or oligosaccharide group. This has been illustrated by thecoupling of N- and O-glycosides to the linkers, which have been used foroligosaccharide synthesis following coupling to the resin. The nature ofthese linkers is such that as well as the potential to immobilise anytype of sugar, any sugar donor can be subsequently used foroligosaccharide synthesis, thereby allowing rapid and efficient couplingprocedures. Suitable sugar donors include, but are not limited toorthoesters, thioorthoesters, cyanoalkylidene derivatives, 1-O-acylsugars, acetimidates, trichloroacetimidates, thioglycosides, glycosylthiocyanates, pentenyl glycosides, pentenoylglycosides, isoprenylglycosides, glycals, tetramethylphosphoro diamidates, sugar diazirines,selenoglycosides, phosphorodithioates, glycosyl-dialkylphosphites,glycosylsulphoxides and glycosylfluorides.

[0076] The stability of the linkers means that orthogonalhydroxy-protecting groups can be employed in sugar protection. Theseprotecting groups include, but are not limited to, acyl-type, trityl,benzyl type, acetal type or various silyl and photolabile protectinggroups.

[0077] The ease of linker synthesis means that the second functionalgroup on the linker may be a halogen, alcohol, thiol or secondary amine,eg.

[0078] X=Halogen, OH, COOH, SH, NHR

[0079] Similarly, the ease of linker synthesis also means that anyfunctionalised resin may be used to immobilise the linker, eg. MBHAresin, amino or carboxy tentagel resins, 4-sulfamylbenzoyl AM resin etc.

[0080] C-Acylation of dimedone with, for example, a mono-protecteddi-carboxylic acid is readily achieved via a carbodiimide activated,DMAP catalysed condensation in dry DCM. Removal of the ester protectionand coupling of the first amino sugar residue generates a sugar-linkerconjugate which can be coupled readily to an amino-functionalised resinsupport via a carbodiimide-mediated condensation. This reaction can bemonitored using conventional amine tests such as TNBS or ninhydrin, toensure quantitative acylation. Alternatively, the linker can besynthesised directly on the resin, followed by introduction of the firstsugar residue on to the linker-resin conjugate. Both methods areillustrated in FIG. 3.

[0081] If an ether-type linkage between the primary sugar residue andthe resin is required, then modification of the linker with4-aminobenzylalcohol to generate the ODmab-type entity allows thismethod of chemical ligation, as illustated in FIG. 4.

[0082] Following selective deprotection of one hydroxyl group, thesecond sugar residue is coupled using any of the sugar donors referredto above, as illustrated in FIG. 8. A portion of the resin is readilycleaved using either ammonia, hydrazine or a primary amine, as shown inFIG. 5, and the cleavage mixture is analysed by TLC to monitor thereaction progress. Completion of the reaction is indicated by thedisappearance of the monosaccharide. The sequential deprotection andcoupling of the following sugar residues is continued until the desiredoligosaccharide is complete, as illustrated in FIG. 1. The protectinggroups are then removed, and the oligosaccharide is cleaved from theresin support using either ammonia, hydrazine, or a primary amine, in asuitable solvent.

[0083] The resin-linker system of the invention is ideal for thesynthesis of combinatorial oligosaccharide libraries , as shown in FIG.2, and for the immobilisation of mono- or oligosaccharides, as shown inFIG. 7.

[0084] The invention will now be described in detail by way of referenceonly to the following non-limiting examples.

EXAMPLES 1-5 Synthesis of a Specially Protected Thioglycoside-Type SugarDonor (FIG. 9)

[0085] 1 Ethyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside

[0086] A mixture of galactose pentaacetate (38.00 g, 97.43 mmol),(ethylthio)trimethylsilane (19.60 g, 146.15 mmol) and trimethylsilyltrifluoromethanesulfonate (23.60 g, 106.20 mmol) in CH₂Cl₂ (150 ml) wasstirred overnight at room temperature. The reaction mixture was dilutedwith CH₂Cl₂ (150 ml) and washed with 1M Na₂CO₃ solution (300 ml), water(300 ml), dried over MgSO₄ and concentrated. The residue wascrystallised from hexane/di-isopropyl ether 1:1 (v/v) to give ethyl2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (34.00 g, 89%).

[0087] Rt 0.43 (hexane/EtOAc 1:1); FAB MS C₁₆H₂₄O₉S (392.3) m/z (%) 415[M+Na]⁺ (100), 393 [M+H]⁺ (20), 331 (56).

[0088] 2 Ethyl 4, 6-O-benzylidene-1-thio-β-D-galacto-pyranoside

[0089] A mixture of ethyl2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (10 g, 25.51 mmol)and sodium methoxide (200 mg, 3.7 mmol) was stirred in abs. MeOH (100ml) at room temperature for 2 hours. The reaction mixture wasneutralised with Amberlite IRA 120 (H+) ion exchange resin andevaporated. The residue was taken up in the (1:?) mixture ofbenzaldehyde/formic acid (21.2 ml) and stirred at room temperature for90 minutes. The reaction mixture was diluted with ether (200 ml) andkept at −15° C. for 2 hours. The precipitate formed was collected andpurified by chromatography using CHCl₃/ethanol 10:3 (v/v) to give ethyl4,6-O-benzylidene-1-thio-β-D-galacto-pyranoside (8.1 g, 64.5%).

[0090] R_(f) 0.64 (CHCl₃/ethanol 10:3).

[0091] 3 Ethyl 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-galactopyranoside

[0092] Ethyl 4,6-O-benzylidene-1-thio-β-D-galacto-pyranoside (6.90 g,22.11 mmol) in 60 ml DMF was added dropwise at 0° C. to a suspension ofsodium hydride 60% (2.65 g, 66.34 mmol) in 60 ml DMF. The mixture wasstirred at room temperature for 1 hour, then benzyl bromide (11.34 g,66.34 mmol) was added dropwise at 0° C. The mixture was stirred at roomtemperature overnight. The mixture was evaporated, and xylene (2×50 ml)was distilled from the residue. The residue was taken up in ether (300ml) and washed with 2×100 ml water. The organic layer was dried overMgSO₄, evaporated and crystallized from MeOH giving ethyl2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (8.90 g,82%).

[0093] R_(f) 0.51 hexane/EtOAc 1:1 v/v); ¹H NMR (CDCl₃) δ7.55-7.25 (m,15H, 15 Ar-H), 5.47 (s, 1H, CHAr), 4.88-4.75 (4d, 4H, 2 CH₂Ar), 4.44 (d,1H, H-1, J_(1.2)=10.89 Hz), 4.30 (dd, 1H, H-6′), 4.16 (d, 1H, H-4),(3.97 (dd, 1H, H-3), 3.88 (t, 1H, H-2), 3.60 (dd, 1H, H-6), 3.35 (d, 1H,H-5), 2.90-2.40 (m, 2H, CH₂S), 1.33 (t, 3H, Me); FAB MS C₂₉H₃₂O₅S(492.40) m/z (%) 515 [M+Na]⁺ (100), 493 [M+H]⁺ (41), 431 (53).

[0094] 4 Ethyl 2,3,6-tri-O-benzyl-1-thio-β-D-galacto-pyranoside

[0095] A mixture of crude ethyl2,3-di-O-benzyl-4,6-O-benzylidene-1-thio-β-D-galactopyranoside (5.4 g,10.97 mmol), sodium cyanoborohydride (6.89 g, 109.7 mmol) and a fewgrains of methyl orange indicator was stirred in THF (60 ml) at 0° C.THF saturated with HCl was added very slowly until a permanent pinkcolour was obtained. The reaction mixture was stirred at roomtemperature for 20 min, then neutralised with dry NH₃ and evaporated.The residue was taken up in CHCl₃ (100 ml), washed with saturated NaHCO₃solution (50 ml), dried over MgSO₄ and evaporated. The residue wasdissolved in MeOH (50 ml), reflux for 10 min and evaporated. The crudeproduct was purified by chromatography using 1,2-dichloroethane/ethylacetate 10:0.5 as the mobile phase to give methyl2,3,6-tri-O-benzyl-1-thio-β-D-galactopyranoside (4.14 g, 75%).

[0096] R_(f) 0.43 (1,2-dichloroethane/EtOAc 10:0.5 v/v); ¹H NMR (CDCl₃)δ7.40-7.26 (m, 15H, 15 Ar-H), 4.88, 4.76, 4.73, 4.71 (4d, 4H, 2 CH₂Ar),4.57 (s, 2H, CH₂Ar), 4.42 (d0.1, H-1, J_(1.2)=9.64 Hz), 4.10 (m, 1H,H-4), (3.76 (dd, 1H, H-3), 3.67 (t, 1H, H-2), 3.55 (m, 2H, H-6), 2.75(m, 2H, CH₂S), 2.50 (bs, 1H, OH), 1.31 (t, 3H, CH₃); FAB MS C₂₉H₃₄O₅S(494.61) m/z (%) 627 [M+Cs]⁺ (70), 517 [M+Na]⁺ (30), 495 [M+H]⁺ (12).

[0097] 5 Ethyl2,3,6-tri-O-benzyl-4-bromoacetyl-1-thio-β-D-galactopyranoside

[0098] A mixture of ethyl2,3,6-tri-O-benzyl-1-thio-β-D-galactopyranoside (4.14 g, 8.38 mmol),sym. collidine (3.65 g, 30.16 mmol), and 4-dimethylaminopyridine in dryCH₂Cl₂ (60 ml) was stirred at 0° C. and bromoacetyl bromide (2.53, 2.57mmol) in CH₂Cl₂ added dropwise in 15 minutes. The reaction mixture wasdiluted with CH₂Cl₂ (100 ml) and washed with 5% HCl solution (3×30 ml)and saturated NaHCO₃ solution (30 ml). The solution was dried over MgSO₄and evaporated. The residue was purified by chromatography usinghexane/EtOAc 2:1 as the mobile phase to give ethyl2,3,6-tri-O-benzyl-4-bromoacetyl-1-thio-β-D-galacto-pyranoside (4.84 g,94%)

[0099]¹H NMR (CDCl₃) δ7.40-7.25 (m, 15H, 15 Ar-H), 4.80-4.50 (m, 6H, 3CH₂Ar), 4.45 (d, 1H, H-1, J_(1.2)=9.53 Hz), 2.73 (m, 2H, CH₂S), 1.30 (t,3H, CH₃); FAB MS C₃₁H₃₅BrO₆S (615.56) m/z (%) 638 [M+Na]⁺ (15), 616[M+H]⁺ (32), 509 (80), 463 (21), 419 (18).

EXAMPLES 6-10 Synthesis of a Partially-Protected Glycosyl Amine (FIG. 9)

[0100] 6 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide

[0101] 1,2,3,4,6-penta-O-acetyl-galactopyranose (1.17 g, 3 mmol) wasdissolved in dry CH₂Cl₂ (15 ml), then trimethylsilyl azide (416 mg, 3.6mmol) and SnCl₄ (0.18 ml) were added under nitrogen. The mixture wasstirred at room temperature for 24 hours. The reaction mixture wassubsequently diluted with CH₂Cl₂ (40 ml), dried over MgSo₄ andevaporated. The residue was purified by chromatography usinghexane/EtOAc 8:7 v/v as the mobile phase to give2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl azide (1.05 g, 94%).

[0102] R_(f) 0.74 (hexane/EtOAc 8:7 v/v); ¹H NMR (CDCl₃) δ5.41 (d, 1H,H-4), 5.17 (t, 1H, H-2), 5.04 (dd, 1H, H-3), 4.60 (d, 1H, H-1,J_(1.2)=10.09 Hz), 4.19 (m, 2H, H-6), 4.00 (m, 1H, H-5), 2.15-1.98 (4s,12H, 4 OAc); FAB MS C₁₄H₁₉N₃O₉ (373.32) m/z (%) 396 [M+Na]⁺ (100), 374[M+H]⁺ (35), 331 (23).

[0103] 7 4,6-O-benzylidene-β-D-galactopyranosyl azide

[0104] A mixture of 2,3,4,6-tetra-O-acetyl-β-D-galacto-pyranosyl azide(19.35 g, 51.79 mmol) and sodium methoxide (200 mg, 3.7 mmol) wasstirred in abs. MeOH (100 ml) at room temperature for 2 hours. Thereaction mixture was neutralised with Amberlite IRA 120 (H+) ionexchange resin and evaporated. The residue was taken up in the mixtureof benzaldehyde/formic acid (1:1) (52 ml) and stirred at roomtemperature for 90 minutes. The reaction mixture was evaporated and theresidue was taken up in ether (60 ml) and kept at −15° C. for 2 hours.The precipitate formed was collected by filtration and dried at roomtemperature affording 4,6-O-benzylidene-β-D-galactopyranosyl azide (11.8g 78%).

[0105] R_(f) 0.64 (CHCl₃/ethanol 10:1.5).

[0106] 8 2,3-di-O-benzyl-4,6-O-benzylidene-β-D-galacto-pyranosyl azide

[0107] 4,6-O-benzylidene-β-D-galactopyranosyl azide (11.8 g, 40.27 mmol)in 60 ml DMF was added dropwise at 0° C. to a suspension of sodiumhydride 60% (6.21 g, 155.38 mmol) in 60 ml DMF. The mixture was stirredat room temperature for 1 hour, then benzyl bromide (26.57 g, 155.38mmol) was added dropwise at 0° C. The mixture was stirred at roomtemperature overnight. The mixture was evaporated, and xylene (2×50 ml)was distilled from the residue. The residue was taken up in ether (500ml) and washed with 2×100 ml water. The organic layer was dried overMgSO₄ and evaporated, giving methyl2,3-di-O-benzyl-4,6-O-benzylidene-β-D-galactopyranosyl azide as a cruderesidue (19.4 g).

[0108] 9 2,3,6-tri-O-benzyl-β-D-galactopyranosyl azide

[0109] A mixture of crude2,3-di-O-benzyl-4,6-O-benzylidene-β-D-galactopyranosyl azide (9.00 g,19.02 mmol), sodium cyanoborohydride (12.00 g, 190.2 mmol) and a fewgrains of methyl orange indicator was stirred in THF (80 ml) at 0° C.THF saturated with HCl was added very slowly until a permanent pinkcolour was obtained. The reaction mixture was stirred at roomtemperature for 20 min, then neutralised with dry NH₃ and evaporated.The residue was taken up in CHCl₃ (100 ml), washed with saturated NaHCO₃solution (50 ml), dried over MgSO₄ and evaporated. The residue wasdissolved in MeOH (50 ml) and kept under reflux for 10 min andevaporated. The crude product was purified by chromatography using1,2-dichloro-ethane/EtOAc 10:0.4 as the mobile phase to give2,3,6-tri-O-benzyl-β-D-galactopyranosyl azide (6.50 g, 72%).

[0110] R_(f) 0.42 (1,2-dichloroethane/EtOAc 10:0.4 v/v); ¹H NMR (CDCl₃)δ7.40 (m, 15H, 15 Ar-H), 4.90-4.55 (m, 6H, 3 CH₂Ar), 4.06 (m, 1H, H-4),(3.82-3.70 (m, 3H, H-3, H-2, H-5), 3.65 (dd, 1H, H-6′), 3.60 (d, 1H,H-1, J_(1.2)=8.64 Hz), 3.51 (dd, 1-H, H-6); FAB MS C₂₇H₂₉N₃O₅ (475.40)m/z (%) 608 [M+Cs]⁺ (10), 498 [M+Na]⁺ (65), 476 [M+H]⁺ (25), 433 (75),341 (20).

[0111] 10 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine

[0112] A mixture of 2,3,6-tri-O-benzyl-β-D-galacto-pyranosyl azide (3.00g, 6.31 mmol), propane-1,3-dithiol (3.40 g, 31.50 mmol), andtriethylamine (3.50 g, 31.5 mmol) in MeOH (31 ml) was stirred undernitrogen at room temperature for 10 hours. The reaction mixture wasevaporated and purified by chromatography using CHCl₃/EtOH 10:0.3 v/v togive 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine (2.66 g, 94%);

[0113] R_(f) 0.38 (CHCl₃/EtOH 10:0.3 v/v); FAB MS C₂₇H₃₁NO₅ (449.33) m/z(%) 472 [M+Na]⁺ (75), 450 [M+H]⁺ (100).

EXAMPLE 11 Synthesis of a Glycosyl Amine—Ddh-Benzyl Ester Conjugate inSolution (FIG. 3)

[0114] 11 N-(Benzyl6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine

[0115] A mixture of benzyl 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate (932 mg, 2.60 mmol),2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine in CH₂Cl₂ (2.0 ml) wasstirred at room temperature for 2 days. The reaction mixture wasevaporated and purified by chromatography using hexane/EtOAc 1:1 as themobile phase to give N-(Benzyl6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate -6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine (1.70 g, 95%);

[0116] R_(f) 0.32 (hexane/EtOAc 1:1 v/v); ¹H NMR (CDCl₃) δ7.37-7.26 (m,5H, 5 Ar-H), 5.40-5.00 (m, 7H, 7 sugar protons), 3.10, 2.85 (2t, 4H, 2CH₂), 2.38 (2s, 4H, Dde 2 CH₂), 2.06-1.98 (4s, 12H, 4 OAc), 1.80 (m, 4H,2 CH₂), 1.02, 1.00 (2s, 6H, Dde 2CH₃); FAB MS C₃₅H₄₅NO₁₃ (687.23) m/z(%) 710 [M+Na]⁺ (35), 688 [M+H]⁺ (100), 356 (60).

EXAMPLE 12 Synthesis of a Fully Protected Glycosyl Amine—Ddh ConjugateDeprotecting a “Fully Protected Amine—DdH Ester Conjugate” in Solution(FIG. 3)

[0117] 12 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine

[0118] N-(Benzyl6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine (1.27 g, 1.84 mmol) washydrogenated over Pd/C (10%) (200 mg) in MeOH (20 ml) at roomtemperature for 10 hours. The catalyst was filtered off, and thefiltrate was evaporated and then chromatographed using CHCl_(3/)MeOH10:0.5 v/v as the mobile phase to giveN-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine 1.10 g, 98%);

[0119] R_(f) 0.38 (CHCl₃/MeOH 10:0.5 v/v); ¹H NMR (CDCl₃) δ5.40-5.00 (m,7H, 7 sugar protons), 3.15, 2.86 (2t, 4H, 2 CH₂), 2.45 (2s, 4H, Dde 2CH₂), 2.10-1.98 (4s, 12H, 4 OAc), 1.80-1.65 (m, 4H, 2 CH₂), 1.02, 1.00(2s, 6H, Dde 2CH₃); FAB MS C₂₈H₃₉NO₁₃ (597.33) m/z (%) 620 [M+Na]+(55),598 [M+H]⁺ (100).

EXAMPLE 13 Synthesis of a Glycosyl Amine—Ddh-Methyl Ester Conjugate inSolution (FIG. 3)

[0120] 13 N-(Methyl6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine

[0121] Reaction 11 was repeated with the difference that methyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate was usedinstead of benzyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate. Yield:92%;

[0122] R_(f) 0.28 (hexane/EtOAc 1:1 v/v) ;FAB MS C₂₉H₄₁NO₁₃ (611.45) m/z(%) 624 [M+Na]⁺ (100), 612 [M+H]⁺ (34)

EXAMPLE 14 Synthesis of a Glycosyl Amine—Ddh-t-Butyl Ester Conjugate inSolution (FIG. 3)

[0123] 14 N-(t-Butyl6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine

[0124] Reaction 11 was repeated with the difference that t-butyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate was usedinstead of benzyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate. Yield:96%;

[0125] R_(f) 0.35 (hexane/EtOAc 1:1 v/v); FAB MS C₃₂H₄₇NO₁₃ (653.37) m/z(%) 676 [M+Na]⁺ (80), 677 [M+H]⁺ (100).

EXAMPLE15 Synthesis of Ddh-OH Benzyl Ester in Solution (FIG. 3)

[0126] 15 Benzyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-hexanoate

[0127] To a stirred solution of mono-benzyl adipate (2.36 g, 10 mmol) indry CH₂Cl₂ (50 ml) was added 5,5-dimethyl-1,3-cyclohexanedione (1.4 g,10 mmol), N,N′-dicyclohexylcarbodiimide (2.1 g, 10.1 mmol) and4-dimethylaminopyridine (1.22 g, 10 mmol). The resulting solution wasallowed to stir at room temperature for 18 h. The solution was cooledand filtered to remove the precipitated N,N′-dicyclohexylurea. Thefiltrate was evaporated and the residue redissolved in EtOAc (50 ml) andwashed with 1 M KHSO₄. The organic extract was washed with brine (92×10ml), dried (MgSO₄) and evaporated to yield a white/yellow amorphouspowder. Flash silica chromatography (EtOAc/hexane 1:2 v/v) affordedbenzyl 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate(3.00 g, 84%) as a white crystalline solid.

[0128]¹H MMR (CDCl₃) δ18.10 (s, 1H, OH), 7.30 (s, 5H, 5Ar-H), 5.06 (s,2H, CH₂Ar), 3.00 (t, 2H, CH₂), 2.47 (s, 2H, Dde CH₂), 2.35 (t, 2H,CH₂CO₂), 2.29 (s, 2H, Dde CH₂), 1.65 (m, 4H, 2 CH₂), 1.01 (s, 6H, 2CH₃); FAB MS C₂₁H₂₆O₅ (358.18) m/z (%) 359 [M+H]⁺ (100), 267 (40); HRMS(FAB) Found: m/z 359.1858 Calcd for C₂₁H₂₇O₅: (M+H), 359.1850.

EXAMPLE 16 Synthesis of Ddh-OH by Deprotection of a Ddh-OH Ester (FIG.3)

[0129] 16 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoicacid

[0130] Benzyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidend)-hexanoate (1.50 g,4.19 mmol) was hydrogenated over Pd/C (10%) (150 mg) in MeOH (20 ml) atroom temperature for 10 hours. The catalyst was filtered off, and thefiltrate was evaporated, yielding6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid (1.10g, 98%);

[0131] R_(f) 0.35 (hexane/EtOAc 2:1 v/v); FAB MS C₁₄H₂₀O₅ (268.12) m/z(%) 313 [M+2Na]⁺ (34), 291 [M+Na]⁺ (100), 269 [M+H]⁺ (16).

EXAMPLE 17 Synthesis of a Ddh-OH Methyl Ester in Solution (FIG. 3)

[0132] 17 Methyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-hexanoate

[0133] Reaction 15 was repeated, with the difference that mono-methyladipate was used instead of mono-benzyl adipate, and afforded methyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate (2.39 g,85%).

[0134] R_(f) 0.32 (EtOAc/hexane 1:2 v/v) FAB MS C₁₅H₂₂O₅ (282.22) m/z(%) 305 [M+H]⁺ (100), 283 [M+H]⁺ (66).

EXAMPLE 18 Synthesis of Ddh-OH t-Butyl Ester in Solution (FIG. 3)

[0135] 18 t-Butyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate

[0136] Reaction 15 was repeated, with the difference that mono-t-butyladipate was used instead of mono-benzyl adipate, and afforded t-butyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate (2.62 g,81%).

[0137] R_(f) 0.36 (EtOAc/hexane 1:2 v/v) FAB MS C₁₈H₂₈O₅ (324.41) m/z(%) 347 [M+H]⁺ (100), 325 [M+H]⁺ (43), 267 (80).

EXAMPLE 19 Synthesis of Ddh-OH by Deprotection of a Ddh-OH t-Butyl Ester(see 16, FIG. 3)

[0138] 19 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoicacid

[0139] t-Butyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate (100 mg,0.30 mmol) was dissolved in CH₂Cl₂/TFA 1:1 mixture (2 ml) and stirred atroom temperature for 1 h. The reaction mixture was evaporated giving6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid (0.81g, 98%)

EXAMPLE 20 Synthesis of Ddh-OH from Cyclic Anhydrides (see 16, FIG. 3)

[0140] 20 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoicacid

[0141] A mixture of glutaric anhydride (2.28 g, 20 mmol), dimedone (2.8g, 20 mmol), 4-dimethylamino-pyridine (3.99 g, 30 mmol) in abs. pyridine(50 ml) was stirred at room temperature for 24 h. The reaction mixturewas evaporated and the residue was taken up in CHCl₃ (100 ml), washed 5%HC1 solution (3×25 ml), saturated NaHCO₃ solution, dried over MgSO₄ andevaporated. The residue was purified by chromatography usingether/acetic acid (10 ml:1 drop) as the mobile phase to give6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid (2.28g, 45%).

EXAMPLE 21 Synthesis of a Fully Protected Glycosyl Amine—Ddh ConjugateUsing Ddh-OH in Solution (See 12, FIG. 3)

[0142] 21 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine

[0143] A mixture of6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid (400mg, 1.49 mmol), 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine (259 mg,0.74 mmol) in abs. EtOH was stirred under reflux for 2 h. The reactionmixture was evaporated and chromatographed using CHCl₃/MeOH 10:0.5 v/vto give N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine(410 mg, 92%).

EXAMPLE 22 Synthesis of a Partially Protected Glycosyl Amine—DdhConjugate Using Ddh-OH in Solution (FIG. 3)

[0144] 22 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine

[0145] Reaction 21 was repeated with the difference that2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine was used instead of2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine, and affordedN-(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoic acid6-yl)2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine (299 mg, 90%).

[0146] R_(f) 0.34 (CHCl₃/MeOH 10:0.1 v/v) FAB MS C₃₇H₄₃NO₇ (613.41) m/z(%) 649 [M+2Na]⁺(34), 626 [M+Na]⁺ (100), 614 [M+H]⁺ (65).

EXAMPLE 23 Synthesis of Ddh-Aminobenzyl Linker in Solution (FIG. 4)

[0147] 23 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 4-amino-benzylalcohol

[0148] Reaction 21 was repeated with the difference that 4-aminobenzylalcohol was used instead of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylamine, and affordedN-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)4-aminobenzyl alcohol (259 mg, 94%).

[0149] R_(f) 0.40 (EtOAc/hexane/acetic acid 2:1:0.1 v/v/v); FAB MSC₂H₂₇NO₅ (373.43) m/z (%) 418 [M+2Na]⁺(24), 396 (M+Na]⁺ (100), 374[M+H]⁺ (35).

EXAMPLE 24 Synthesis of Ddh-Aminobenzyl t-Butyl Ester Linker in Solution(FIG. 4)

[0150] 24 N-(L-Butyl 6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl) 4-aminobenzyl alcohol

[0151] A mixture of t-butyl 6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate (400 mg, 1.23 mmol) and4-aminobenzyl alcohol (605 mg, 4.92 mmol) in abs. EtOH was stirred underreflux for 2 h. The reaction mixture was evaporated and purified bychromatography using CHCl₃/MeOH 9:1 as the mobile phase to giveN-(t-Butyl 6-(4,4-dimethyl -2,6-dioxocyclohexylidene)-hexanoate-6-yl)4-aminobenzyl alcohol (395 mg, 75%)

[0152] R_(f) 0.52 (CHCl₃/MeOH 9:1 v/v) FAB MS C₂₅H₃₅NO₅ (429.53) m/z (%)452 [M+Na]⁺ (100), 430 [M+H]⁺ (32), 372 (64).

EXAMPLE 25 Synthesis of Ddh-Aminobenzyl t-Butyl EsterTrichloroacetimidate Activated Linker in Solution (FIG. 4)

[0153] 25 N-(t-Butyl 6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl) 4-aminobenzyltrichloroacetimidate

[0154] A mixture of N-(t-butyl 6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate-6-yl) 4-aminobenzyl alcohol (500mg, 1.16 mmol) and trichloroacetonitrile (503 mg, 3.49 mmol) in CH₂Cl₂(5 ml) was stirred at 0° C. and 1,8-diazabicyclo(5.4.0)undec-7-ene (5mg, 0.03 mmol) added. The reaction mixture was stirred at 0° C. for 90minutes, at room temperature for 2 h, then evaporated. The residue waspurified by chromatography using EtOAc/hexane 1:1 as the mobile phase togive N-(t-butyl 6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate-6-yl) 4-aminobenzyltrichloroacetimidate (580 mg, 87%);

[0155] R_(f) 0.41 (EtOAc/hexane 1:1 v/v); FAB MS C₂₇H₃₅Cl₃N₂O₅ (573.94)m/z (%) 595 [M+Na]⁺ (100), 753 [M+H]⁺ (40), 515 (39), 430 (54).

EXAMPLE 26 Synthesis of a Fully Protected Sugar (Sugar-Linker Bond isnot at the Glycosidic Position)—Ddh-Aminobenzyl t-Butyl Ester ConjugateVia Trichloroacetimidate Activation (FIG. 4)

[0156] 26 Benzyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-[N-(t-butyl 6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)4-aminobenzyl]-α-D-glucopyranoside

[0157] N-(t-Butyl 6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl) 4-aminobenzyltrichloro-acetimidate (400 mg, 0.70 mmol) was added at 20° C. undernitrogen to a solution of Benzyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-α-D-glucopyranoside (155 mg, 0.35 mmol) in CH₂Cl₂ (6ml). Trifluoromethanesulphonic acid in ether (0.1 M, 0.06 ml) was addedand the mixture was stirred for 30 min at 20° C. The reaction wasstopped with 5% NaHCO₃ solution (0.25 ml). After filtration of themixture and evaporation of the filtrate, the crude residue was purifiedby chromatography using EtOAc/hexane 2:1 v/v as the mobile phase to giveBenzyl 2-acetamido-3-O-acetyl-6-O-benzyl -2-deoxy-4-O-[N-(t-butyl6-(4,4-dimethyl -2,6-dioxocyclo-hexylidene)-hexanoate-6-yl)4-aminobenzyl]-α-D-gluco-pyranoside (210 mg, 71%).

[0158] R_(f) 0.37 (EtOAc/hexane 2:1 v/v); FAB MS C₄₉H₆₂N₂O₁₁ (855.01)m/z (%) 877 [M+Na]⁺ (100), 855 [M+H]⁺ (35), 797 (73).

EXAMPLE 27 Synthesis of a Fully Protected Glycoside (Sugar-Linker Bondat the Glycosidic Position)—Ddh-Aminobenzyl Linker—Resin ViaTrichloroacetimidate Activation (FIG. 4)

[0159] 27 [N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 4-aminobenzyl] 2,3,4,6tetra-O-acetyl-β-D-glucopyranoside MBHAresin conjugate

[0160] N-(t-Butyl 6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate-6-yl) 4-aminobenzyltrichloro-acetimidate (400 mg, 0.70 mmol) was added at 20° C. undernitrogen to a solution of 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose (121mg, 0.35 mmol) in CH₂Cl₂ (6 ml). Trifluoromethanesulphonic acid in ether(0.1 M, 0.06 ml) was added and the mixture was stirred for 30 min at 20°C. The reaction was stopped with 5% NaHCO₃ solution (0.25 ml) Afterfiltration of the mixture, the filtrate was evaporated. The unpurifiedresidue was taken up in CH₂Cl_(2/)TFA mixture (1:1) (5 ml), stirred atroom temperature for 1 h and evaporated. The resulting acid wasdissolved in CH₂Cl₂ (5 ml), N,N′-diisopropylcarbodiimide (128 mg, 1mmol) added, and the mixture was gently agitated with MBHA resin (100mg)(swelled in DMF for 20 min.) for 30 min.

EXAMPLE 28 Synthesis of a Fully Protected Glycoside (Sugar—Linker Bondis at the Glycoside Position)—Ddh-Aminobenzyl Benzyl Ester Conjugate ViaDMTST Promoted Glycosylation (see 26, FIG. 4)

[0161] 28 [N-[Benzyl(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoate]-6-yl4-aminobenzyl]-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside

[0162] A mixture of N-[Benzyl(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate]-6-yl 4-aminobenzylalcohol (500 mg, 1.08 mmol), methyl2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranoside (400 mg, 1.08 mmol) inCH₂Cl₂ (10 ml) was stirred at room temperature and DMTST (835 mg, 3.24mmol) added. The solution was stirred at room temperature for 1 h andwashed with saturated NaHCO₃ solution (3 ml), dried over MgSO₄ andevaporated. The residue was purified by chromatography usinghexane/EtOAc 1:1 v/v as the mobile phase to give [N-[Benzyl(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate]-6-yl4-aminobenzyl]-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (610 mg, 75%).

[0163] R_(f) 0.47 (hexane/EtOAc 1:1 v/v); FAB MS C₄₂H₅₁NO₁₄ (793.83) m/z(%) 816 [M+Na]⁺ (100), 794 [M+H]⁺ (25), 702 (66).

EXAMPLE 29 Synthesis of a Fully Protected Glycoside (Sugar-Linker Bondis at the Glycosidic Position)—Ddh-Aminobenzyl Linker—Resin ConjugateVia DIPCDI Activation (see 27, FIG. 4)

[0164] 29 [N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 4-aminobenzyl]-2,3,4,6-tetra-O-acetyl-β-D-glucopyranosideMBHA resin conjugate

[0165] [N-[Benzyl(6-(4,4-dimethyl-2,6-dioxocylo-hexylidene)-hexanoate]-6-yl4-aminobenzyl]-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (500 mg, 0.63mmol) was hydrogenated over Pd/C (10%) (200 mg) in MeOH (20 ml) at roomtemperature for 10 hours. The catalyst was filtered off and the filtratewas evaporated. The residue was dissolved in CH₂Cl₂ (5 ml),N,N′-diisopropylcarbodiimide (128 mg, 1 mmol) added, and the mixture wasgently agitated with MBHA resin (200 mg) (pre-swelled in DMF for 20min.) for 30 min.

EXAMPLE 30 Synthesis of a Partially Protected Glycosyl Amine—DdhConjugate Using Ddh-OH t-Butyl Ester in Solution (see 22, FIG. 3)

[0166] 30 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine

[0167] A mixture of t-butyl6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate (400 mg,1.23 mmol) and 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine (276 mg,0.61 mmol) in abs. EtOH (10 ml) was stirred under reflux for 2 h. Thereaction mixture was evaporated. The residue was taken up in CH₂Cl₂/TFAmixture (1:1) (10 ml) and stirred at room temperature for 1 h. Thereaction mixture was evaporated and purified by chromatography usingCHCl₃/MeOH 10:0.1 v/v as the mobile phase to giveN-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine (280 mg, 73%).

[0168] R_(f) 0.34 (CHCl₃/MeOH 10:0.1 v/v) FAB MS C₃₇H₄₃NO₇ (613.41) m/z(%) 649 [M+2Na]⁺(34), 626 [M+Na]⁺ (100), 614 [M+H]⁺ (65).

EXAMPLE 31 Synthesis of a Fully Protected Glycosyl Amine—Ddh—ResinConjugate Where the Resin Coupling is the Final Step (FIG. 3)

[0169] 31 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine—MBHAconjugate

[0170] MBHA resin (Subst. ratio: 0.42 mmol/g) (200 mg) bearing a totalamine functionality of 0.084 mmol was swollen in DMF for 20 min. Theresin was then washed with fresh DMF andN-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)2,3,4,6-tetra-O-acetyl-β-D-gluco-pyranosyl amine (200 mg, 4 equiv.) andN,N′-diisopropyl-carbodiimide (53 μl,4 equiv.) were added in DMF (5 ml)and the resin gently agitated for 30 min. The TNBS test was faintlypositive so using the above conditions, a double coupling was performed,this time producing a negative TNBS test result. The resin was washedwith DMF, methanol and finally ether. The resin was then allowed to dryin vacuum over KOH overnight.

EXAMPLE 32 Synthesis of a Fully Protected Sugar (Sugar—Linker Bond isNot at the Glycosidic Position)—Ddh—Resin Conjugate Where the ResinCoupling is the Final Step (see 27, FIG. 4)

[0171] 32 Benzyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-[N-(6-(4,4-dimethyl-2,6-dioxocyclohexyl-idene)-hexanoic acid-6-yl)4-aminobenzyl]-α-D-glucopyranoside—MBHA resin conjugate

[0172] Benzyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy -4-O-[N-(t-butyl6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate -6-yl)4-aminobenzyl]-α-D-glucopyranoside (290 mg, 0.33 mmol) was dissolved inCH₂Cl₂/TFA mixture (1:1) and stirred at room temperature for 1 h. Thereaction mixture was evaporated, and procedure 31 was used to bind thecompound to the MBHA resin.

EXAMPLE 33 Synthesis of Ddh-Aminobenzyl Linker—Resin Conjugate WithSelective Resin Coupling (Unprotected Hydroxyl Group is Present on theLinker) (FIG. 10)

[0173] 33 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 4-amino-benzylalcohol—MBHA resin conjugate

[0174] MBHA resin (100 mg) bearing a total amine functionality of 0.042mmol was swelled in DMF for 20 min. The resin was then washed with freshDMF and N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)4-aminobenzyl alcohol (63 mg, 4 equiv.) and1-isogutyloxy-carbonyl-2-isobutyloxy-1,2-dihydroquinoline (EEDQ) (51 mg,4 equiv.) were added in DMF (5 ml) and the resin gently agitated for 24h. The TNBS test was faintly positive so using the above conditions, adouble coupling was performed, this time producing a negative TNBS testresult. The resin was washed with DMF (5×10 ml).

EXAMPLE 34 Synthesis of Ddh-Aminobenzyl Trichloroacetimidate ActivatedLinker—Resin Conjugate When the Activation Takes Place on the Resin(FIG. 10)

[0175] 34 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoate -6-yl)4-aminobenzyl trichloroacetimidate—MBHA resin conjugate

[0176] Resin from Example 33 was treated with trichloroacetonitrile (50mg, 0.33 mmol) in CH₂Cl₂ (1 ml) was stirred at 0° C. and1,8-diazabicyclo(5.4.0)undec-7-ene (1 mg, 0.003 mmol) added. Thereaction mixture was stirred at 0° C. for 90 minutes, at roomtemperature for 2 h, then the resin was filtered off and washed with DMF(5×10 ml).

EXAMPLE 35 Synthesis of a Fully Protected Sugar (Sugar—Linker Bond isNot at the Glycosidic Position)—Ddh—Resin Conjugate When the SugarCoupling is the Final Step (see 32, FIG. 4)

[0177] 35 Benzyl 2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-[N-(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoic acid-6-yl)4-aminobenzyl]-α-D-glucopyranoside—MBHA resin conjugate

[0178] Resin from Example 34 was added at room temperature to a solutionof Benzyl 2-acetamido-3-O-acetyl -6-O-benzyl-2-deoxy-α-D-glucopyranoside(75 mg, 0.16 mmol) in CH₂Cl₂ (1 ml). Trifluoromethanesulphonic acid inether (0.1 M, 60 μl) was added and the mixture was stirred for 30 min.The reaction was stopped with triethylamine (120 μl) and washed with DMF(5×10 ml).

EXAMPLE 36 First Step of the Solid Phase Synthesis of the Resin—Ddh- orDdH-Aminobenzyl—Linker (FIG. 3)

[0179] 36 Adipic acid—MBHA resin conjugate

[0180] MBHA resin (1.0 g) bearing a total amine functionality of 0.42mmol was swelled in DMF for 20 min. The resin was then treated with amixture of adipic acid (1.41 g, 10 mmol) andN,N′-diisopropylcarbodiimide in CH₂Cl₂ (10 ml) for 60 min. A secondcoupling was performed in DMF to get a negative ninhydrin test. Theresin was washed with DMF (5×10 ml).

EXAMPLE 37 Second Step of the Solid Phase Synthesis of the Resin—Ddh- orDdH-Aminobenzyl—Linker (FIG. 3)

[0181] 37 6-Hydroxy-6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoicacid-MBHA resin conjugate

[0182] To the resin from Example 36 a mixture of5,5-dimethyl-1,3-cyclohexanedione (280 mg, 2.0 mmol),N,N′-dicyclohexylcarbodiimide (283 mg, 2.00 mmol) and4-dimethylaminopyridine (244 mg, 2.00 mmol) was added in CH₂Cl₂ (10 ml)and stirred at room temperature for 18 h. The resin was washed with DMF(5×10 ml).

EXAMPLE 38 Solid Phase Synthesis of a Fully Protected GlycosylAmine—Ddh—Resin Conjugate (see 31, FIG. 3)

[0183] 38 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine—MBHA resinconjugate

[0184] The resin from Example 37 was reacted with2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine (712 mg, 2.00 mmol) inDMF (5 ml) at room temperature for 2 days. The resin was washed with DMF(5×10 ml).

EXAMPLE 39 Solid Phase Synthesis of a Partially Protected GlycosylAmine—Ddh—Resin Conjugate (FIG. 3)

[0185] 39 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine—MBHA resinconjugate

[0186] The resin from Example 37 was reacted with2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine (900 mg, 2.00 mmol) inabs. EtOH under reflux for 2 h. The resin was washed with DMF (5×10 ml).

EXAMPLE 40 Solid Phase Synthesis of Ddh-Aminobenzyl Linker—ResinConjugate (see 33, FIG. 10)

[0187] 40 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) 4-amino-benzylalcohol—MBHA resin conjugate

[0188] A mixture of resin from Example 37 and 4-aminobenzyl alcohol (246mg, 2.00 mmol) in abs. EtOH was stirred under reflux for 2 h, thenwashed with DMF (5×10 ml).

EXAMPLE 41 Cleavage of a Fully Protected Glycosyl Amine—Ddh—ResinConjugate Affording Fully Protected Glycosyl Amine (FIG. 11)

[0189] 41 Cleavage ofN-(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoic acid-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine—MBHA resin conjugate byNH₃ treatment.

[0190] Resin from Example 38 (10 mg) was treated with saturated NH₃/MeOHsolution (0.2 ml) at room temperature for 5 min. The resin was filteredoff, the filtrate was evaporated, giving2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine in quantitative yield.

EXAMPLE 42 Cleavage of a Fully Protected Glycosyl Amine—Ddh—ResinConjugate Affording Fully Protected Reducing Sugar

[0191] 42 Cleavage ofN-(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoic acid-6-yl)2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl amine—MBHA resin conjugate byNH₃ treatment, affording a reducing carbohydrate derivative (FIG. 11).

[0192] Resin from Example 38 (10 mg) was treated with saturated NH₃/MeOHsolution (0.2 ml) at room temperature for 5 min. The resin was filteredoff, the filtrate was evaporated. The residue was dissolved in themixture of acetone/water 10:1 v/v (0.2 ml), acidified with acetic acid(20 μl ) and stirred at room temperature for 1 h. The solution wasevaporated giving 2,3,4,6-tetra-O-acetyl-β-D-glucopyranose inquantitative yield.

EXAMPLE 43 Carbohydrate Deprotection of the Fully Protected Sugar—DdhLinker—Resin Conjugate (FIG. 12)

[0193] 43 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) β-D-glucopyranosyl amine—MBHA resin conjugate

[0194] The resin from Example 38 was gently agitated with sodiummethoxide (200 mg, 3.70 mmol) in abs. MeOH (5 ml) at room temperaturefor 1 h. The resin was washed with abs. MeOH (5×10 ml), DMF(5×10 ml),ether (5×10 ml) and dried under high vacuum for 1 h, giving theresin-bonded unprotected β-D-glucopyranosyl amine. A sample of resin (5mg) was cleaved by NH₃/MeOH (Example 41), and the resulting product wasanalyzed by TLC and mass spectometry, proving the quantitativedeprotection.

EXAMPLE 44 Synthesis of a Library of Di-, Tri- and Tetrasaccharides on aSolid Support (FIG. 12)

[0195] 44 A mixture of mono-, di- and tri-O-(2,3,4-tri-O-benzylα,β-L-fucopyranosyl) (1→2), (1→3), (1→6),(1→6)-[N-(6-(4,4-dimethyl-2,6-dioxocyclo-hexylidene)-hexanoicacid-6-yl)]β-D-glucopyranosyl amine—MBHA resin conjugate

[0196] A mixture of resin from Example 43 and ethyl2,3,4-tri-O-benzyl-1-thio-β-L-fucopyranoside (950 mg, 2 mmol) in dryCH₂Cl₂ (10 ml) was treated with dimethyl-(methylthio)-sulphoniumtrifluoromethanesulphonate (DMTST) (1.50 g, 5.81 mmol) at roomtemperature for 1 h. The resin was washed with dry CH₂Cl₂ (5×10 ml).

EXAMPLE 45 Cleavage of a Library of Di-, Tri- and Tetrasaccharides fromthe Resin Affording Glycosyl Amine of Oligosaccharides (FIG. 12)

[0197] 45 A mixture of mono-, di- and tri-O-(2,3,4-tri-O-benzylα,β-L-fucopyranosyl) (1→2), (1→3), (1→4), (1→6)-β-D-glucopyranosyl amine

[0198] The resin from Example 44 was treated with NH₃/MeOH (10 ml) for 5min. The resin was filtered off, and the filtrate was evaporated givinga mixture of disaccharides, trisaccharides, and tetresaccharides. FAB MSdisaccharides C₃₃H₄₁NO₉ (595.66), trisaccharides C₆₀H₆₉NO₁₃ (1012.16),tetrasaccharides C₈₇H₉₇NO₁₇ (1429.66) (m/z (%) 618 [M_(di)+Na]⁺ (41),596 [M_(di)+H]⁺ (57), 1034 [M_(tri)+Na]⁺ (56), 1012 [M_(tri)+H]⁺ (100),1450 [M_(tetra)+Na]⁺ (8), 1428 [M_(tetra)+H]⁺ (10).

EXAMPLE 46 Synthesis of a Second Sugar—Glycosyl Amine—Ddh Linker—ResinConjugate (FIG. 13)

[0199] 46 O-(2,3,6-tri-O-benzyl-4-O-bromoacetyl-α,β-D-galactopyranosyl)(1→4)-[N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl)] 2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine—MBHA resinconjugate

[0200] A mixture of resin from Example 39 and ethyl2,3,6-tri-O-benzyl-4-O-bromoacetyl-1-thio-β-D-galacto-pyranoside (1.25g, 2 mmol) in dry CH₂Cl₂ (10 ml) was treated withdimethyl(methylthio)sulphonium trifluoro-methanesulphonate (DMTST) (1.50g, 5.81 mmol) at room temperature for 1 h. The resin was washed with dryCH₂Cl₂ (5×10 ml).

EXAMPLE 47 Selective Deprotection of the Second Sugar —GlycosylAmine—Ddh Linker—Resin Conjugate (FIG. 13)

[0201] 47 O-(2,3,6-tri-O-benzyl-α,β-D-galacto-pyranosyl)(1→4)-[N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid-6-yl)]2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine—MBHA resin conjugate

[0202] The resin from Example 46 was gently agitated with sodiummethoxide (200 mg, 3.70 mmol) in abs. MeOH (5 ml) at room temperaturefor 1 h. The resin was washed with abs. MeOH (5×10 ml), DMF(5×10 ml),ether (5×10 ml) and dried under high vacuum for 1 h, giving the resinbonded partially unprotected disaccharide A sample of resin (5 mg) wascleaved by NH₃/MeOH (Example 41) and the resulting product was analyzedby TLC and mass spectometry, proving the quantitative deprotection.

EXAMPLE 48 Cleavage of a Second Sugar—Glycosyl Amine —Ddh Linker ResinConjugate Affording a Glycosyl Amine of a Disaccharide (FIG. 13)

[0203] 48 O-(2,3,6-tri-O-benzyl -α,β,-D-galacto-pyranosyl) (1→4)-2,3,6-tri-O-benzyl-β-D-galactopyranosyl amine

[0204] The resin from Example 47 was treated with NH₃/MeOH (10 ml) for 5min. The resin was filtered off, and the filtrate was evaporated givingan anomeric mixture of disaccharides. FAB MS C54H₅₉NO₁₀ (882.01) (m/z(%) 904 [M+Na]⁺ (100), 880 [M+H]⁺ (41).

EXAMPLE 49 Cleavage of a Carbohydrate—Ddh—Aminobenzyl Linker—ResinConjugate Affording an Aminobenzyl Protected Carbohydrate (FIG. 14)

[0205] 49 4-aminobenzyl β-D-glucopyranoside

[0206] The resin from Example 29 was treated with NH₃/MeOH (5 ml)overnight. The resin was filtered off, and the filtrate was evaporatedgiving 4-aminobenzyl β-D-glucopyranoside.

[0207] R_(f) 0.55 (CHCl₃/MeOH/H₂O 10:4:0.5 v/v/v); FAB MS C₁₃H₁₉NO₅(269.28) m/z (%) 402 [M+Cs]⁺ (25), 292 [M+Na]⁺ (50), 270 [M+H]⁺ (18).

EXAMPLE 50 Deprotection of 4-Aminobenzyl Protected Carbohydrate (FIG.14)

[0208] 50 β-D-Glucopyranose

[0209] 4-Aminobenzyl β-D-glucopyranoside (110 mg, 0.40 mmol) washydrogenated over Pd/C (10%) (50 mg) in MeOH (5 ml) at room temperaturefor 4 hours. The catalyst was filtered off and the filtrate wasevaporated affording D-glucose in quantitative yield.

EXAMPLE 51 Immobilization of an Oligosaccharide (FIG. 15)

[0210] 51 O-[O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl (1→4))-2,3,6-tri-O-acetyl-β-D-clucopyranosyl (1→4)]-2,3,6-tri-O-acetyl-β-D-glucopyranosyl amine using6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid—MBHAresin conjugate

[0211] The resin from Example 37 was reacted withO-[O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl(1→4))-2,3,6-tri-O-acetyl-β-D-glucopyranosyl(1→4)]-2,3,6-tri-O-acetyl-β-D-glucopyranosylamine (1.80 g, 2.00 mmol) in DMF (5 ml) at room temperature for 2 days.The resin was washed with DMF (5×10 ml).

EXAMPLE 52 Synthesis of an Aminosugar—Ddh—Resin Conjugate (FIG. 16)

[0212] 52 N-(6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoicacid-6-yl) D-glucosamine—MBHA resin conjugate

[0213] A mixture of resin from Example 37 and glucosamine (350 mg, 2mmol) in DMF (20 ml) was stirred at room temperature for 2 days. Theresin was filtered off, washed with DMF/H₂O 4:1 (5×10 ml), DMF 5×10 ml,MeOH (5×10), ether (5×10 ml), and dried under high vacuum overnight.

[0214] It will be apparent to the person skilled in the art that whilethe invention has been described in some detail for the purposes ofclarity and understanding, various modifications and alterations to theembodiments and methods described herein may be made without departingfrom the scope of the inventive concept disclosed in this invention.

[0215] References cited herein are listed on the following pages, andare incorporated by this reference.

[0216] References

[0217] Adinolfi, M., Barone, G., De Napoli, L., Iadonisi, A. andPiccialli, G. Tetrahedron Lett., 1996 37 5007.

[0218] Bannwarth, W., Huebscher, J. and Barner, R. Bioorganic and Med.Chem. Lett., 1996 6 1525.

[0219] Boren, T. et al. Science, 1993 262 1892.

[0220] Bycroft, B. W., Chan, W. C., Chhabra, S. R. and Hone, N. D. J.Chem. Soc., Chem. Commun., 1993 778.

[0221] Chan, W. C., Bycroft, B. W., Evans, D. J. and White, P. D. J.Chem. Soc., Chem. Commun., 1995 2209.

[0222] Douglas, S. P., Whitfield, D. M. and Krepinsky, J. J. J. Am.Chem. Soc., 1995, 117 2116.

[0223] Fisher, J. F. et al. J. Med. Chem., 1991 34 3140.

[0224] Frechet, J. M. and Schuerch, C. J. Am. Chem. Soc., 1971 93 492.

[0225] Frechet, J. M. and Schuerch. C. J. Am. Chem. Soc., 1972 94 604.

[0226] Gambaryan, A. S. et al. FEBS Lett., 1995 366 57.

[0227] Guthrie, R. D., Jenkins, A. D. and Stehlicek, J. J. Am. Chem.Soc., 1971 (c) 2690.

[0228] Guthrie, R. D., Jenkins, A. D. and Roberts, J. A. F. J. Chem.Soc., Perkin Trans. 1, 1973 1 2441.

[0229] Lasky, L. A. Science, 1992 258 964.

[0230] Lee, Y. C. in Carbohydrate Recognition in Cellular Function (GBlock and S. Harnett, Eds.), John Wiley & Sons, 1989 80.

[0231] Merrifield, R. B. J. Am. Chem. Soc., 1963 85 2149.

[0232] Rademann, J. and Schmidt, R. R. Tetrahedron Lett., 1996 37 3989.

[0233] Randolph, J. T., McClure, K. F. and Danishefsky, S. J. J. Am.Chem. Soc., 1995 117 5712.

[0234] Roberge, J. Y., Beebe, X. and Danishefsky, S. J. Science, 1995269 202.

[0235] Rodriguez, R. E. et al. Neurosci. Lett., 1989 101 89.

[0236] Varki, A. Glycobiology, 1993 3 97.

[0237] Yan, L., Taylor, C. M., Goodnow Jr., R. and Kahne, D. J. Am.Chem. Soc., 1994 116 6953.

1. A support for solid-phase synthesis of oligosaccharides, said supportcomprising a) a resin, b) a linker covalently attached to the resin, andc) one or more saccharide groups covalently attached to the resin viathe linker, wherein the linker is a 2-substituted-1,3-dioxocycloalkanecompound, and said support having general formula I

in which R¹ and R² may be the same or different, and is each hydrogen orC₁₋₄ alkyl; preferably both R¹ and R² are methyl; R′ is an amino sugar,a glycosylamine, or a glycosylamine of an oligosaccharide; a mono oroligosaccharide coupled through an alkyl-, substituted alkyl-, aryl-,substituted aryl-, cycloalkyl-, or substituted cycloalkyl-amino group;or a mono or oligosaccharide coupled through a carboxyalkyl-,substituted carboxyalkyl-, carboxyaryl-, substituted carboxyaryl-,carboxycycloalkyl-, or substituted carboxycycloalkyl-amino group, and R″is an alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, orsubstituted cycloalkyl spacer group which is directly coupled to theresin support, or which may optionally be coupled to the resin supportvia a covalent linkage which is stable to conditions of oligosaccharidesynthesis and cleavage.
 2. A support according to claim 1, in which bothR¹ and R² are methyl.
 3. A support according to claim 1 or claim 2, inwhich R′ is an oligosaccharide-O—CH₂—(C₆H₄)—NH,monosaccharide-O—CH₂—(C₆H₄)—NH, amino-oligosaccharide-C O₂CH₂—(C₆H₄)NH,or amino-monosaccharide-CO₂CH₂—(C₆H₄)—NH group.
 4. A support accordingto any one of claims 1 to 3, in which the covalent linkage to the resinis provided by a —CONH—, —O—, —S—, —COO—, —CH—N—, —NHCONH—, —NHCSNH, or—NHNH— grouping.
 5. A support according to any one of claims 1 to 4, inwhich the linker is functionalised Dde, Ddh or ODMab.
 6. A supportaccording to any one of claims 1 to 5, comprising a resin, a linker anda monosaccharide, an oligosaccharide, an aminosaccharide or anamino-oligosaccharide.
 7. A support for solid-phase synthesis comprisinga resin and a linker group, wherein the linker is a2-substituted-1,3-dioxocycloalkane of general formula II:

in which X is OH or NH₂; R¹ and R² may be the same or different, and iseach hydrogen or C₁₋₄ alkyl; and R″ is an alkyl, substituted alkyl,aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl spacergroup which is directly coupled to the resin support, or which mayoptionally be coupled to the resin support via a covalent linkage whichis stable to conditions of oligosaccharide synthesis and cleavage.
 8. Asupport according to claim 7, in which R¹ and R² are both methyl
 9. Asupport according to claim 7 or claim 8, in which the covalent linkageto the resin is provided by a —CONH—, —O—, —S—, —COO—, —CH═N—, —NHCONH—,—NHCSNH, or —NHNH— grouping.
 10. A linker-saccharide complex in whichthe linker group is as defined in claim 1 or claim 2 and the saccharideis as defined in claim 1 or claim
 6. 11. A compound carrying functionalgroups suitable to attach a primary amine to a resin via covalent bondswhich are stable to conditions of oligosaccharide synthesis andcleavage, said compound having general formula III

in which X is OH or NH₂; R¹ and R² may be the same or different, and iseach hydrogen or C₁₋₄ alkyl, and R″ is an alkyl, substituted alkyl,aryl, substituted aryl, cycloalkyl, or substituted cycloalkyl spacergroup, which carries a functionality capable of reacting with afunctionalised resin.
 12. A compound according to claim 11, in whichboth R¹ and R² are methyl.
 13. A compound according to claim 11 or claim12, in which the functionality on R″ is a carboxyl group.
 14. A compoundaccording to claim 11, which is6-hydroxy-6-(4,4-dimethyl-2,6-dioxocyclohexylidene)-hexanoic acid or anester thereof.
 15. A compound according to claim 14, in which the esteris a benzyl, methyl or t-butyl ester.
 16. A support according to any oneof claims 1 to 6, in which the linker is a compound according to any oneof claims 11 to
 15. 17. A support according to any one of claims 7 to 9,in which the linker is a compound according to any one of claims 11 to15.
 18. A linker-saccharide complex according to claim 10, in which thelinker is a compound according to any one of claims 11 to
 15. 19. A kitfor solid phase synthesis or combinatorial synthesis ofoligosaccharides, comprising: a) a resin-linker-saccharide supportaccording to any one of claims 1 to 5 or 16, b) a linker-saccharidecomplex according to claims 10 or 17, or c) a resin-linker supportaccording to any one of claims 7 to 17, and optionally also comprisingone or more protecting agents, deprotecting agents, and/or solventssuitable for solid phase or combinatorial synthesis.
 20. A method ofsolid-phase synthesis of oligosaccharides, comprising the step ofsequentially linking mono- or oligosaccharide groups to a support asdefined in any one of claims 1 to 9 or
 16. 21. A method of synthesis ofa linker group according to general formula I as defined in claim 1,comprising the step of C-acylation of a 2-substituted1,3-dioxocyclohexane compound with a dicarboxylic acid, and optionallyreacting the product of the C-acylation reaction with 4-aminobenzylalcohol, to form the 4-aminobenzyl derivative.
 22. A method according toclaim 21, in which the dicarboxylic acid is mono-protected by esterformation.
 23. A method according to claim 21 or claim 22, in which theC-acylation reaction is activated with carbodiimide and catalysed byN,N′-dimethylaminopyridine.
 24. A method of synthesis of a resin-linkersupport according to any one of claims 6 to 9, comprising the step ofswelling a resin in a suitable solvent, treating the swollen resin witha dicarboxylic acid, and reacting the thus-produced product with a2-substituted 1,3-dioxocycloalkane compound.
 25. A method according toany one of claims 21 to 24, in which the 2-substituted1,3-dioxocycloalkane compound is 5,5-dimethyl-1,3-cyclohexanedione. 26.A method according to any one of claims 21 to 25, in which thedicarboxylic acid is adipic acid.